Goetz, Matthew P.

The Oncology Grand Rounds series is designed to place original reports published in the Journal into clinical context. A case presentation is followed by a description of diagnostic and management challenges, a review of the relevant literature, and a summary of the authors’ suggested management approaches. The goal of this series is to help readers better understand how to apply the results of key studies, including those published in Journal of Clinical Oncology, to patients seen in their own clinical practice.

Abstract Triple Negative Breast Cancer (TNBC) affects approximately 15-20% of BC patients, yet accounts for a disproportionately higher rate of BC morbidity and mortality, in part due to lack of targeted therapies. We have shown that estrogen receptor beta (ERβ) is expressed in approximately 20% of TN breast tumors and that ligand-mediated activation of ERβ with estradiol (E2) or ERβ-selective agonists decreases tumor cell proliferation, invasion and migration in vitro and in vivo. Therefore, we aimed to elucidate the mechanisms by which ERβ elicits its anti-cancer effects in TNBC. RNAseq analysis of ERβ-expressing MDA-MB-231 cells demonstrated that ERβ significantly downregulates NFκB signaling in the presence of E2. ChIPseq for ERβ in these cells revealed that ERβ primarily associated with estrogen response elements, but 12% of all ERβ binding sites were located at NFκB consensus motifs. Using an NFκB reporter construct and qPCR, ERβ was shown to block TNFα-mediated induction of NFκB signaling and NFκB target gene expression. RNAseq analysis of MDA-MB-231-ERβ cells treated with TNFα or E2+TNFα revealed substantial global inhibition of TNFα regulated genes in the presence of E2. ChIPseq for NFκB demonstrated that ERβ significantly alters NFκB’s cistrome whereby it can both diminish NFκB binding and redistribute NFκB throughout the genome. ChIPseq also demonstrated that ligand-mediated activation of ERβ significantly diminished an activating histone mark (H3K27Ac) at many of these NFκB target genes while enhancing a repressive mark (H3K27Me3). The addition of H3K27Me3 at these loci was shown to occur through the recruitment of the histone methyltransferase, EZH2. Drug-mediated blockade of EZH2 activity reversed suppression of NFκB target gene expression by ERβ. Knockdown of NFκB or Mutation of ERβ’s DNA binding domain rendered ERβ incapable of associating with DNA, recruiting EZH2, methylating NFκB target gene loci, repressing NFκB target gene expression and inhibiting proliferation. Interestingly, ERβ was shown to elicit more potent anti-cancer effects in TNBC cells expression a constitutively active form of NFκB. These finding suggest that a primary mechanism by which ERβ functions as a tumor suppressor is through inhibition of NFκB pathway activity. Our studies have also revealed that ERβ functions as a molecular switch for EZH2 and repurposes it for tumor suppressive activities, as EZH2 has previously been reported to enrich NFκB signaling in TNBC. These findings could address the paradox that high EZH2 expression is associated with worse TNBC patient outcomes, while high H3K27Me3 expression is associated with improved patient outcomes. Currently, a Mayo Clinic Breast Cancer SPORE prospective phase II clinical trial is underway to investigate the efficacy of estradiol for the treatment of metastatic ERβ+TNBC and to further evaluate the cross-talk between ERβ, EZH2 and NFκB signaling.

PURPOSE Cancer-related cognitive impairment (CRCI) is common during adjuvant chemotherapy and may persist. TAILORx provided a novel opportunity to prospectively assess patient-reported cognitive impairment among women with early breast cancer who were randomly assigned to chemoendocrine therapy (CT+E) versus endocrine therapy alone (E), allowing us to quantify the unique contribution of chemotherapy to CRCI. METHODS Women with a 21-gene recurrence score of 11 to 25 enrolled in TAILORX were randomly assigned to CT+E or E. Cognitive impairment was assessed among a subgroup of 552 evaluable women using the 37-item Functional Assessment of Cancer Therapy-Cognitive Function (FACT-Cog) questionnaire, administered at baseline, 3, 6, 12, 24, and 36 months. The FACT-Cog included the 20-item Perceived Cognitive Impairment (PCI) scale, our primary end point. Clinically meaningful changes were defined a priori and linear regression was used to model PCI scores on baseline PCI, treatment, and other factors. RESULTS FACT-Cog PCI scores were significantly lower, indicating more impairment, at 3, 6, 12, 24, and 36 months compared with baseline for both groups. The magnitude of PCI change scores was greater for CT+E than E at 3 months, the prespecified primary trial end point, and at 6 months, but not at 12, 24, and 36 months. Tests of an interaction between menopausal status and treatment were nonsignificant. CONCLUSION Adjuvant CT+E is associated with significantly greater CRCI compared with E at 3 and 6 months. These differences abated over time, with no significant differences observed at 12 months and beyond. These findings indicate that chemotherapy produces early, but not sustained, cognitive impairment relative to E, providing reassurance to patients and clinicians in whom adjuvant chemotherapy is indicated to reduce recurrence risk.

Several new systemic therapy options have become available for patients with metastatic breast cancer, which have led to improvements in survival. In addition to patient and clinical factors, the treatment selection primarily depends on the tumor biology (hormone-receptor status and HER2-status). The NCCN Guidelines specific to the workup and treatment of patients with recurrent/stage IV breast cancer are discussed in this article.

Abstract Background: Endx is a Tam metabolite with promising antitumor activity in Tam and aromatase inhibitor (AI) resistant estrogen receptor (ER) positive (+) metastatic breast cancer (MBC). Methods: This randomized phase II study compared progression-free survival (PFS) and toxicity of Endx 80 mg/day to Tam 20 mg/day in patients (pts) with ER+ MBC. Eligibility included postmenopausal women, ECOG PS 0-2, prior progression on AI but not Tam (unlimited endocrine therapy [ET] lines allowed), and a preregistration biopsy confirming ER+ (>10% nuclear staining) and HER2-negative MBC. Stratified randomization was used balancing prior CDK 4/6 inhibitor (CDK 4/6i) use and/or everolimus (yes/no), measurable disease (yes/no) and endocrine resistance (primary/secondary) between arms. Pts randomized to Tam were allowed to cross over to Endx at progression. Due to short expected median PFS, differences in PFS were assessed using approaches for interval-censored data (ICD). 40 eligible pts were to be randomized to each arm so a one-sided alpha=0.10 generalized log-rank test (GLRT) would have a 90% chance of detecting a 50% decrease in hazard of disease progression with Endx (median: 6 months) relative to Tam (median: 3 months). Secondary endpoints include clinical benefit rate (stable or partial response > 6 cycles) (CBR) for measurable and non-measurable disease. Pharmacokinetic (PK) data were obtained Day (d) 1 (4 hour), end of cycle 2, and at time of progression. Results: From March 2015 to March 2017, 108 women with endocrine refractory recurrent or MBC were preregistered. 27 pts did not register due to: biopsy demonstrating ER-/HER2- (3 pts), ER+/HER2+ (5 pts), cancer other than breast (3 pts), no cancer in specimen (6 pts), brain metastases (2 pts), acute infection (1 pt), progression on or recent use of Tam (2 pts), or pt refusal (5 pts). 4/81 pts who registered were excluded due to ineligibility (3 pts) or refusal to start protocol treatment (1 pt). The study cohort consisted of 40 pts randomized to Endx and 37 pts to Tam. The median (m) number of ETs in the metastatic setting was 2 (range 1-4) for each arm including prior CDK 4/6i (Endx: 42.5%, Tam: 29.7%) and everolimus (Endx: 35.0%, Tam: 40.5%). The m cycle number was 6 for Endx (range: 1-35) and 3 for Tam (range: 1-42). PFS for Endx was not significantly different compared to Tam (HR= 0.77; 95% CI: 0.49-1.22, GLRT p=0.309; mPFS: Endx 130 days [95% CI: 76-138 days] and Tam 42 days [95%CI: 24-129 days]). However, PFS was significantly longer in pts with no prior CDK 4/6i in the Endx arm (GLRT p=0.002; HR(no/yes)=0.31; 95%CI: 0.15-0.65) but not in the Tam arm (GLRT p=0.708) (unplanned analysis). Severe (grade (G) 3+) toxicities included: Endx G3 hypertriglyceridemia (3 pts); Tam: G3 hypertension with G2 stroke (1 pt), G3 thromboembolic event (1 pt), and G3 abdominal, bone and liver pain (1 pt). In Endx arm, d1 m Endx plasma concentration (conc) was 216 ng/ml (n=17; range: 144-400). For Tam arm, d1 m Tam conc was 17 ng/ml (n=17; range:11-23) (Endx not detectable). For the 25 pts that crossed over to Endx, CBR was 28.0% (90% CI: 14.0-46.2%) and 14 pts had pk data at progression. A lower median Endx conc (6 ng/ml range: 3.3-16.3) was observed in Tam patients at progression who then had Endx clinical benefit compared to Tam pts without clinical benefit after Endx crossover (median Endx 12 ng/ml; range 4.4-36.6).Conclusions: In endocrine-resistant breast cancer, Z-Endx was not significantly superior to Tam, but clinical benefit was observed in 28% that crossed over to Endx after Tam progression. In pts with no prior CDK 4/6i, the observation of significantly longer PFS in the Endx arm is hypothesis generating. Support: U10CA180821, U10CA180882, U24CA196171, U10CA180820 (ECOG-ACRIN), https://acknowledgments.alliancefound.org; Clinical Trials.gov Identifier:NCT02311933 Citation Format: Matthew P. Goetz, Vera J Suman, Joel M Reid, Mary Kuffel, Sarah A Buhrow, Renee M McGovern, John Black, Travis Dockter, William F Symmans, Minetta C Liu, John R Hawse, James Doroshow, Anna M Storniolo, Jerry M Collins, Howard Streicher, Matthew M Ames, James N Ingle, Ann Partridge, Lisa Carey. A randomized phase II trial of tamoxifen versus Z-endoxifen HCL in postmenopausal women with metastatic estrogen receptor positive, HER2 negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD7-06.

Abstract Background: Combination treatments of CDK4 & 6 inhibitors (CDK4 & 6i) with endocrine therapy (ET) have improved outcomes in patients with HR-positive (HR+), HER2-negative (HER2-) advanced breast cancer (ABC). Aside from estrogen receptor positivity (ER+), predictive biomarkers of clinical benefit from CDK4 & 6i in combination with ET remain elusive. We assessed clinical outcomes by genomic alterations detected in baseline circulating tumor DNA (ctDNA) from patients treated with abemaciclib plus a nonsteroidal aromatase inhibitor (NSAI) versus placebo plus NSAI in the Phase 3 study MONARCH 3 (NCT02246621). Methods: MONARCH 3 randomized 493 postmenopausal women with HR+, HER2- ABC who had no prior systemic therapy in the advanced setting to treatment with abemaciclib plus NSAI or placebo plus NSAI. Biomarker analysis of baseline ctDNA was an exploratory objective; plasma was analyzed by the Guardant360 next-generation sequencing (NGS)-based assay to identify potential tumor-related (i.e., somatic) genomic alterations including point mutations (SNV), indels, amplifications, and fusions in over 70 cancer-related genes. Genomic alterations detected in baseline ctDNA were associated with clinical outcomes including progression-free survival (PFS) and objective response rate (ORR). Results: Baseline ctDNA results were available for 295 patients (201 abemaciclib; 94 placebo) with 83% of patients harboring one or more detectable genomic alterations. Commonly altered genes of interest (% frequency) detected in baseline ctDNA included: PIK3CA (38%); TP53 (26%); EGFR (15%); FGFR1 (12%); NF1 (12%); MYC (9%); CCND1 (9%); ESR1 (5%).Overall, patients treated with placebo plus NSAI who harbored detectable ctDNA genomic alterations had shorter median PFS compared to patients without detectable genomic alterations, as shown in the Table (14.9 months [95% CI: 11.0-23.1] vs 19.2 months [95% CI 9.4-NR]). Moreover, genomic alterations in EGFR, FGFR1, MYC, and CCND1 were associated with median PFS <12 months with placebo plus NSAI. However, consistent with the intent-to-treat population, the addition of abemaciclib to NSAI benefitted these genomic subgroups, regardless of ctDNA gene alterations. Additional genomic results, including alterations in cell cycle-associated genes, will be presented. Conclusions: In this exploratory subgroup analysis, the presence of detectable ctDNA genomic alterations was associated with shorter PFS with placebo plus NSAI. Consistently, abemaciclib added to NSAI improved outcomes for genomically identified subgroups. In contrast to prior studies, there was no subgroup (e.g., patients harboring FGFR1 alterations) that did not derive benefit from abemaciclib, supporting the efficacy of abemaciclib, including in difficult to treat tumors. These findings are hypothesis-generating and warrant further investigations in clinical studies of CDK4 & 6i in combination with ET. TableAbemaciclib + NSAIPlacebo + NSAIEvents, n/NMedian PFS (95% CI)Events, n/NMedian PFS (95% CI)Hazard Ratio (95% CI)OVERALLITT population170 / 32828.2 (23.7 - 33.9)123 / 16514.8 (11.2 - 19.2)0.52 (0.42 - 0.66)TR population93 / 20138.7 (31.1 - NR)71 / 9416.5 (11.7 - 23.1)0.45 (0.33 - 0.61)Any Gene AlterationDetected83 / 16634.1 (27.2 - 40.0)62 / 7914.9 (11.0 - 23.1)0.47 (0.34 - 0.66)Not detected10 / 35NR (36.4 - NR)9 / 1519.2 (9.40 - NR)0.31 (0.13 - 0.78)TP53Detected29 / 5327.0 (14.1 - NR)19 / 2315.4 (5.7 - 30.4)0.53 (0.30 - 0.95)Not detected64 / 14839.9 (34.1 - NR)52 / 7117.5 (11.7 - 24.2)0.42 (0.29 - 0.60)EGFRDetected10 / 26NR (32.4 - NR)16 / 1710.9 (2.1 - 24.7)0.20 (0.09 - 0.47)Not detected83 / 17536.4 (27.7 - NR)55 / 7717.6 (14.6 - 25.5)0.52 (0.37 - 0.73)FGFR1Detected15 / 2532.8 (10.1 - NR)10 / 117.6 (1.9 - 15.4)0.37 (0.16 - 0.85)Not detected78 / 17639.9 (31.1 - NR)61 / 8319.2 (14.6 - 26.5)0.45 (0.32 - 0.63)NF1Detected10 / 2435.9 (27 - NR)8 / 1014.6 (1.6 - 33.4)0.33 (0.13 - 0.84)Not detected83 / 17738.7 (30.8 - NR)63 / 8417.5 (11.7 - 24.2)0.47 (0.33 - 0.65)MYCDetected11 / 1720.1 (5.5 - NR)9 / 106.5 (1.2 - 15.7)0.33 (0.13 - 0.86)Not detected82 / 18438.9 (32.9 - NR)62 / 8419.2 (14.6 - 26.5)0.45 (0.32 - 0.63)CCND1Detected8 / 1632.8 (5.5 - NR)10 / 107.2 (3.6 - 9.0)0.28 (0.10 - 0.77)Not detected85 / 18538.7 (31.1 - NR)61 / 8417.6 (14.6 - 25.5)0.48 (0.34 - 0.67)n = number of patients with events; N = total number of patients in the corresponding population and treatment arm; NR = not reached. TR population consists of patients in the ITT population from whom a valid baseline ctDNA result has been obtained. Citation Format: Matthew P Goetz, J. Thaddeus Beck, Mario Campone, Sara Hurvitz, Seock-Ah Im, Stephen Johnston, Antonio Llombart-Cussac, Miguel Martin, Joohyuk Sohn, Masakazu Toi, Lacey M Litchfield, Hillary T Graham, Hong Wang, Sameera R Wijayawardana, Valerie M Jansen, Angelo Di Leo. Efficacy of abemaciclib based on genomic alterations detected in baseline circulating tumor DNA from the MONARCH 3 study of abemaciclib plus nonsteroidal aromatase inhibitor [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD2-06.

Abstract Background: TNBC is the most aggressive sub-type of breast cancer and has limited targeted therapies. Atezolizumab was recently FDA-approved for patients with advanced PD-L1+ TNBC. In a well-characterized cohort of patients with early-stage TNBC, we used the FDA-approved PD-L1 SP142 companion assay for atezolizumab to score whole tumor sections for PD-L1 expression. We determined the frequency and scoring distribution of PD-L1 in these tumors and evaluated PD-L1 status for associations with clinicopathologic variables and outcomes. Methods: Whole tumor sections from 498 women with surgically-treated, non-(distant) metastatic TNBC (ER/PR <1% & HER2-negative by central review) were semi-quantitatively scored for % of PD-L1-staining tumor cells (TC) or tumor-associated immune cells (IC) (PD-L1 SP142, Ventana, performed in CAP/CLIA-certified clinical laboratory). Per FDA assay guidelines, PD-L1+ tumors were defined as ≥1% of tumor-associated immune cells (IC) staining per tumor area. Baseline clinical characteristics, histopathologic features, Ki-67 proliferative indices (PI), and stromal tumor infiltrating lymphocytes (TILs) were correlated with PD-L1 status. The impact of tumoral PD-L1 status on invasive disease-free survival (IDFS) and overall survival (OS) was assessed using Cox proportional hazards models. Results: The majority of tumors were pT1-T2 (95%), pN0-1 (85%), and grade 3 (90%) with Ki-67 >15% (80%). 228/498 (46%) tumors were PD-L1+, distributed as IC score 1-5%: 21%, 6-10%: 13%, 11-20%: 5% and >20%: 7%. 270/498 (54%) were PD-L1-. 43 (8.6%) of tumors had PD-L1 staining in TC (all but 1 were also IC+). PD-L1 IC expression was associated with larger tumor size (p = 0.005), higher nodal stage (≥pN1: 43% vs. 31%, p < 0.001), higher grade (grade 3: 95% vs. 86%, p< 0.001), higher Ki-67 PI (>15%: 88% vs. 69%, p <0.001), and stromal TILs (median score: 40% vs. 10%; p < 0.001). PD-L1 IC status was not associated with patient age. Among histologic subtypes, PD-L1+ tumors were more frequently medullary type (31% vs. 6%) and less frequently metaplastic type (4% vs 10%). At time of this analysis, median follow-up for IDFS and OS were 11 years and 7.2 years, respectively. By univariable analysis, absence of PD-L1 IC expression was associated with poorer IDFS (median: 15.5 vs. 20 years, p = 0.04) and OS (9.5 vs. 17.5 years, p < 0.05).By multivariable analysis, tumor size, stromal TILs, nodal status, and adjuvant chemotherapy (CTX) were significantly associated with IDFS. Age, stromal TILs, nodal status, and receipt of adjuvant CTX were associated with OS. PD-L1+ tumors trended toward improved IDFS (statistical significance not reached), HR: 0.68 (95% CI: 0.45-1.03, p= 0.068) but not OS: 0.90 (95% CI: 0.52-1.57, p= 0.32). Conclusion: Using the PD-L1 SP142 companion assay, almost half of early-stage TNBC were PD-L1+ (IC). However, PD-L1 expression correlated with increasing tumor size and nodal burden, as well as histologic grade, Ki-67 PI, stromal TILs and lymphocyte-enriched subtypes. PD-L1 was associated with improved IDFS and OS in univariable analysis but not multivariable analysis. The majority of the PD-L1+ tumors (73%) had IC scores ≤10%, with heterogeneous tumoral staining. Given the low (1%) assay threshold for positivity, these data emphasize the utility of evaluating PD-L1 in whole tumor sections rather than tissue cores. Citation Format: Jodi M Carter, Mei-Yin C Polley, Jason P Sinnwell, Roberto A Leon-Ferre, Fergus J Couch, Krishna R Kalari, Judy C Boughey, Minetta C Liu, Daniel W Visscher, Matthew P Goetz. Frequency, characteristics and prognostic factors of PD-L1+ triple negative breast cancer using the PD-L1 SP142 companion assay [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD1-08.

Abstract Background: Epigenetic based therapies, such as histone deacetylase inhibitors, have proven successful in many cancer types including lymphomas and myelomas with ongoing phase III clinical trials in NSCLC. Epigenetic modulation in triple negative breast cancer (TNBC) has been increasingly explored in the past decade. A major issue preventing the uptake of these drugs in the clinic is the relatively high doses necessary to achieve therapeutic benefit. It is therefore of interest to identify combinatorial approaches that result in drug de-escalation and enhanced therapeutic benefit. We and others have demonstrated that estrogen receptor beta (ERβ) is expressed in approximately 20% of TNBC patients. ERβ targeted therapies, including estradiol and multiple ERβ selective agonists, result in suppression of cell proliferation, invasion, and migration in vitro and in vivo (Reese, PNAS 2018). For these reasons, we sought to determine if ERβ targeted therapies would synergize with epigenetic based therapies for the treatment of TNBC. Methods: Using multiple TNBC cell lines representative of ERβ+ and ERβ- disease, as well as a model of acquired resistance to ERβ targeted therapies, we performed an epigenetic drug screen assessing 145 compounds alone and in combination with estradiol treatment. The top hit from this screen was extensively profiled across multiple cell lines in the presence and absence of ERβ targeting agents and was compared to the efficacy observed in ERα+ models of breast cancer using cell proliferation assays, RT-PCR, and western blotting. Results: The most significant compound to elicit differential and therapeutically beneficial effects in ERβ+ TNBC was CUDC-101, a multitarget pan-HDACi, EGFRi, and HER2i that was developed based on the structures of Vorinostat, Erlotinib, and Lapatinib. CUDC-101 potently inhibits the growth of TNBC cells and synergizes with E2 stimulation in ERβ+ MDA-MB-231 TNBC cells, but not in ERβ- cells. CUDC-101 was also effective in ERβ-resistant MDA-MB-231 cells and restored the growth inhibitory effects of estrogen in this model. Treatment with Vorinostat (SAHA) alone elicited a similar, though far less potent, synergistic effect in the MDA-MB-231-ERβ+ cell line, suggesting that histone deacetylase inhibition is a critical, but not completely sufficient, component of the synergistic phenotype. CUDC-101, while effective at reducing the viability of MDA-MB-468-ERβ+ cell line, had no synergistic effect with E2 in this model suggesting differences in response between TNBC sub-types. Additionally, CUDC-101 was found to be ineffective in models of ERα+ disease (MCF7 and MCF7-ERβ) in both the presence and absence of E2 treatment. Conclusions: Therapeutic options remain extremely limited for TNBC patients and the development of novel treatment strategies for these patients continue to represent a major unmet clinical need. Here, we present evidence that CUDC-101 elicits potent anti-cancer effects in multiple models of TNBC and synergizes with ERβ-targeted therapies in some models. Our findings further strengthen the body of evidence suggesting ERβ as a valid therapeutic target and important biomarker in TNBC and further implicate HDACi/EGFRi as relevant therapeutic strategies within this patient population. Citation Format: Michael Justin Emch, David Benjamin, Elizabeth Bruinsma, Molly Nelson-Holte, Matthew Goetz, John Hawse. CUDC-101 enhances estrogen-mediated anticancer effects in ERβ+ TNBC [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-05-10.

Abstract Background: Triple Negative Breast Cancer (TNBC) affects approximately 15-20% of BC patients, yet accounts for a disproportionately higher rate of BC morbidity and mortality, in part due to lack of targeted therapies. Using well-validated antibodies, we have found that estrogen receptor beta (ERβ) protein is expressed in approximately 20% of TN breast tumors. We have demonstrated that ligand-mediated activation of ERβ with estradiol (E2) or ERβ-selective agonists decreases tumor cell proliferation, invasion, and migration in vitro, as well as primary tumor growth and metastatic spread in vivo. As such, we aimed to elucidate the mechanisms by which ERβ elicits its anti-cancer effects in TNBC. Methods: RNAseq and ChIPseq were used to identify ERβ and NFκB regulomes and cistromes in TNBC cells. The global effects of ERβ on H3K27Ac and H3K27Me3 were also assessed via ChIPseq. The influence of ERβ on NFκB signaling was determined using co-immunoprecipitation, luciferase assays transcriptional readouts and ChIPseq. The involvement of EZH2 in mediating ERβ’s suppression of NFκB signaling and cancer cell phenotypes was determined using pharmacological approaches. Results: Pathway analysis of ERβ-regulated genes identified NFκB and inflammatory signaling as being highly suppressed in response to E2 treatment. Numerous NFκB target genes were among the most down-regulated genes by ERβ. ChIPseq revealed that ERβ primarily associated with estrogen response elements (EREs), but was also highly enriched at NFκB binding sites following E2 treatment. However, ERβ had no impact on TNFα-mediated NFκB phosphorylation or nuclear localization, but was shown to physically associate with NFκB protein. Using an NFκB reporter construct and qPCR, ERβ was shown to block TNFα-mediated induction of NFκB signaling and NFκB target gene expression. Globally, RNAseq identified 373 genes to be significantly regulated by TNFα in TNBC cells, of which 107 were blocked in the presence of E2+TNFα. Interestingly, E2+TNFα treatment induced novel NFκB binding sites compared to TNFα alone as identified by ChIPseq for NFκB. ChIPseq also demonstrated that ligand-mediated activation of ERβ significantly diminished an activating histone mark (H3K27Ac) at many of these NFκB target genes while enhancing a repressive mark (H3K27Me3). These modifications were shown to occur through the recruitment of the histone methyltransferase, EZH2, to enhancer elements of these NFκB target genes. Drug-mediated blockade of EZH2 activity reversed suppression of NFκB target gene expression by ERβ. Finally, knockdown of NFκB expression inhibited the anti-cancer effects of ERβ while expression of a constitutively active form of NFκB enhanced ERβ’s inhibitory effects. Conclusions: Our data suggest that ERβ elicits its anti-cancer effects, in part, via formation of a novel co-repressor complex consisting of ERβ, NFκB, and EZH2. These data are in keeping with prior observations of the importance of NFκB signaling as it relates to TNBC cell proliferation and invasion, and that decreased expression of NFκB target genes is associated with improved outcomes in TNBC patients. Currently, a Mayo Breast SPORE prospective study is underway to investigate the efficacy of estradiol for the treatment of metastatic ERβ+TNBC and to further evaluate the cross-talk between ERβ and NFκB signaling. Citation Format: Kirsten GM Aspros, Adam W Nelson, Zhenqing Ye, Zhifu Sun, Igor Chernukhin, Jason S Carroll, James N Ingle, Matthew P Goetz, John R Hawse. Estrogen receptor beta repurposes EZH2 to inhibit oncogenic NFκB signaling in triple negative breast cancer [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-05-02.

Abstract Despite the availability of effective drugs that target ERα-positive breast cancer, ERα-positive disease still accounts for more breast cancer related deaths than any other subtype. Relapse in these patients is largely due to the development of resistance to anti-estrogen therapies such as tamoxifen. While tamoxifen and its resistance mechanisms have been extensively studied, relatively little is known about its active metabolite endoxifen. Our group has provided evidence that endoxifen is a potent and clinically relevant metabolite of tamoxifen. Therefore, characterization of endoxifen resistance mechanisms may be crucial to understanding tamoxifen resistance. We have developed novel endoxifen resistant MCF7 and T47D cell lines through chronic exposure to endoxifen over a period of 12-24 months. Using these models and their respective controls, we compared global gene expression profiles of endoxifen resistant cells to tamoxifen resistant cells and found marked differences between the two models. We subjected endoxifen resistant cells to a genome-wide, CRISPR-mediated knockout screen to identify genes involved in mediating endoxifen resistance. Subsequent analyses revealed that disruption of genes regulated by the glucocorticoid receptor (GR) enhanced cells’ ability to survive and proliferate in the presence of endoxifen, suggesting that GR signaling blocks growth of resistant cells. We provide evidence that GR activation via dexamethasone significantly inhibits the proliferation rates of endoxifen resistant cells by 50-60%, with little to no inhibitory effects in endoxifen sensitive models. Follow-up studies suggested that alpha zinc-glycoprotein 1 (AZGP1), a secreted lipolytic enzyme induced by GR, plays a role in mediating this phenotype. AZGP1 RNA and protein are highly expressed in normal breast tissue, but corresponding levels are lower in ERα-positive breast malignancies. In these tumors, low AZGP1 expression correlates with worse prognosis. Additionally, high expression of AZGP1 is associated with improved outcomes in patients with prostate, pancreatic, gastric and breast cancer. AZGP1 is expressed in endocrine sensitive MCF7 and T47D cells, but is silenced in endoxifen resistant models at the mRNA and protein level. Further, treatment of resistant cells with conditioned medium containing AZGP1 resulted in significant inhibition of cell proliferation and migration, and antibody-mediated depletion of AZGP1 from conditioned medium completely reversed these effects. While there are no reports regarding the molecular functions of AZGP1 in cancer we provide evidence that AZGP1 directly inhibits oncogenic signaling in endocrine resistant breast cancer through interaction with, and inhibition of, β6 integrin. Through the analysis of endoxifen resistant breast cancer cells, we have identified novel roles for GR, AZGP1 and β6 integrin in the development and progression of endocrine resistant breast cancer. We demonstrate that activation of GR may elicit therapeutic benefit in endocrine resistant breast cancer, specifically in tumors expressing AZGP1 and β6 integrin. Further, AZGP1 and β6 integrin may represent novel prognostic and/or predictive biomarkers in endocrine resistant disease. Citation Format: Calley J. Jones, Santiago Acero Bedoya, Anna V. Mykytyn, Matthew P. Goetz, John R. Hawse. Glucocorticoid receptor signaling elicits anti-cancer effects in endocrine resistant breast cancer via induction of AZGP1 [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-04-16.